An ABA-serotonin module regulates root suberization and salinity tolerance.

Suberin in roots acts as a physical barrier preventing water/mineral losses. In Arabidopsis, root suberization is regulated by abscisic acid (ABA) and ethylene in response to nutrient stresses. ABA also mediates coordination between microbiota and root endodermis in mineral nutrient homeostasis. However, it is not known whether this regulatory system is common to plants in general, and whether there are other key molecule(s) involved. We show that serotonin acts downstream of ABA in regulating suberization in rice and Arabidopsis and negatively regulates suberization in rice roots in response to salinity. We show that ABA represses transcription of the key gene (OsT5H) in serotonin biosynthesis, thus promoting root suberization in rice. Conversely, overexpression of OsT5H or supplementation with exogenous serotonin represses suberization and reduces tolerance to salt stress. These results identify an ABA-serotonin regulatory module controlling root suberization in rice and Arabidopsis, which is likely to represent a general mechanism as ABA and serotonin are ubiquitous in plants. These findings are of significant importance to breeding novel crop varieties that are resilient to abiotic stresses and developing strategies for production of suberin-rich roots to sequestrate more CO2 , helping to mitigate the effects of climate change.

Three Diverse Granule Preparation Methods for Proteomic Analysis of Mature Rice (Oryza sativa L.) Starch Grain.

Starch is the primary form of reserve carbohydrate storage in plants. Rice (Oryza sativa L.) is a monocot whose reserve starch is organized into compounded structures within the amyloplast, rather than a simple starch grain (SG). The mechanism governing the assembly of the compound SG from polyhedral granules in apposition, however, remains unknown. To further characterize the proteome associated with these compounded structures, three distinct methods of starch granule preparation (dispersion, microsieve, and flotation) were performed. Phase separation of peptides (aqueous trypsin-shaving and isopropanol solubilization of residual peptides) isolated starch granule-associated proteins (SGAPs) from the distal proteome of the amyloplast and the proximal 'amylome' (the amyloplastic proteome), respectively. The term 'distal proteome' refers to SGAPs loosely tethered to the amyloplast, ones that can be rapidly proteolyzed, while proximal SGAPs are those found closer to the remnant amyloplast membrane fragments, perhaps embedded therein-ones that need isopropanol solvent to be removed from the mature organelle surface. These two rice starch-associated peptide samples were analyzed using nano-liquid chromatography-tandem mass spectrometry (Nano-HPLC-MS/MS). Known and novel proteins, as well as septum-like structure (SLS) proteins, in the mature rice SG were found. Data mining and gene ontology software were used to categorize these putative plastoskeletal components as a variety of structural elements, including actins, tubulins, tubulin-like proteins, and cementitious elements such as reticulata related-like (RER) proteins, tegument proteins, and lectins. Delineating the plastoskeletal proteome begins by understanding how each starch granule isolation procedure affects observed cytoplasmic and plastid proteins. The three methods described herein show how the technique used to isolate SGs differentially impacts the subsequent proteomic analysis and results obtained. It can thus be concluded that future investigations must make judicious decisions regarding the methodology used in extracting proteomic information from the compound starch granules being assessed, since different methods are shown to yield contrasting results herein. Data are available via ProteomeXchange with identifier PXD032314.

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support.

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm phenotyping can be used to classify genotypes based on SG morphotype using scanning electron microscopic (SEM) analysis. SGs can be visualized using SEM by slicing through the kernel (pericarp, aleurone layers, and endosperm) and exposing the organellar contents. Current methods require the rice kernel to be embedded in plastic resin and sectioned using a microtome or embedded in a truncated pipette tip and sectioned by hand using a razor blade. The former method requires specialized equipment and is time-consuming, while the latter introduces a new host of problems depending on rice genotype. Chalky rice varieties, particularly, pose a problem for this type of sectioning due to the friable nature of their endosperm tissue. Presented here is a technique for preparing translucent and chalky rice kernel sections for microscopy, requiring only pipette tips and a scalpel blade. Preparing the sections within the confines of a pipette tip prevents rice kernel endosperm from shattering (for translucent or 'vitreous' phenotypes) and crumbling (for chalky phenotypes). Using this technique, endosperm cell patterning and the structure of intact SGs can be observed.

Review of Methodological Approaches to Human Milk Small Extracellular Vesicle Proteomics.

Proteomics can map extracellular vesicles (EVs), including exosomes, across disease states between organisms and cell types. Due to the diverse origin and cargo of EVs, tailoring methodological and analytical techniques can support the reproducibility of results. Proteomics scans are sensitive to in-sample contaminants, which can be retained during EV isolation procedures. Contaminants can also arise from the biological origin of exosomes, such as the lipid-rich environment in human milk. Human milk (HM) EVs and exosomes are emerging as a research interest in health and disease, though the experimental characterization and functional assays remain varied. Past studies of HM EV proteomes have used data-dependent acquisition methods for protein detection, however, improvements in data independent acquisition could allow for previously undetected EV proteins to be identified by mass spectrometry. Depending on the research question, only a specific population of proteins can be compared and measured using isotope and other labelling techniques. In this review, we summarize published HM EV proteomics protocols and suggest a methodological workflow with the end-goal of effective and reproducible analysis of human milk EV proteomes.

Roles of MicroRNA across Prenatal and Postnatal Periods.

Communication between mother and offspring in mammals starts at implantation via the maternal-placental-fetal axis, and continues postpartum via milk targeted to the intestinal mucosa. MicroRNAs (miRNAs), short, noncoding single-stranded RNAs, of about 22 nucleotides in length, are actively involved in many developmental and physiological processes. Here we highlight the role of miRNA in the dynamic signaling that guides infant development, starting from implantation of conceptus and persisting through the prenatal and postnatal periods. miRNAs in body fluids, particularly in amniotic fluid, umbilical cord blood, and breast milk may offer new opportunities to investigate physiological and/or pathological molecular mechanisms that portend to open novel research avenues for the identification of noninvasive biomarkers.

Ingested soluble CD14 contributes to the functional pool of circulating sCD14 in mice.

Soluble CD14 (sCD14) is a pattern recognition receptor and Toll-like co-receptor observed in human milk (5-26µg/mL) and other bodily fluids such as blood (3µg/mL). The most well defined role of sCD14 is to recognize lipopolysaccharide of Gram-negative bacteria and signal an immune response through Toll-like receptor 4 (TLR4). Previous research has shown ingested sCD14 to transfer from the gastrointestinal tract and into the blood stream in neonatal rats. The contribution of human milk sCD14 to circulating levels in the infant and the functionality of the protein, however, remained unknown. Using CD14(-/-) mouse pups fostered to wild type (WT) mothers expressing sCD14 in their milk, we show herein that ingestion of sCD14 resulted in blood sCD14 levels up 0.16±0.09µg/mL. This represents almost one-third (26.7%) of the circulating sCD14 observed in WT pups fostered to WT mothers (0.60±0.14µg/mL). We also demonstrate that ingested-sCD14 transferred to the blood remains functional in its ability to recognize lipopolysaccharide as demonstrated by a significant increase in immune response (IL-6 and TNF-α) in CD14(-/-) pups fostered to WT mothers in comparison to control animals (P=0.002 and P=0.007, respectively). Using human intestinal cells (Caco-2), we also observed a significant decrease in sCD14 transcytosis when TLR4 was knocked down (P<0.001), suggesting sCD14 transfer involves TLR4. The bioavailability of human milk sCD14 established in this report confirms the importance of human milk proteins for the infant and demonstrates the need to improve infant formulas which are lacking in immune proteins such as sCD14.

Ingested soluble CD14 from milk is transferred intact into the blood of newborn rats.

BACKGROUND: Milk acts as an edible immune system that is transferred from mother to newborn. Soluble Cluster of Differentiation 14 (sCD14) is a protein found in significant quantities in human milk (~8-29 µg/ml). At a 10-fold lower concentration in the blood (~3 µg/ml), the most notable role of sCD14 is to sequester lipopolysaccharides of Gram-negative bacteria from immune cells. METHODS: To explore the pharmacodynamics of this milk protein and its biological fate, the biodistribution of radiolabeled sCD14 ((14)C, (125)I) was monitored in 10-d-old rat pups. RESULTS: Up to 3.4 ± 2.2% of the radiolabeled sCD14 administered was observed, intact, in the pup blood for up to 8 h post-ingestion. Additionally, 30.3 ± 13.0% of the radiolabeled sCD14 administered was observed degraded in the stomach at 8 h post-ingestion. A reservoir of intact, administered sCD14 (3.2 ± 0.3%), however, remained in the stomach at 8 h post-ingestion. Intact sCD14 was observed in the small intestine at 5.5 ± 1.6% of the dose fed at 8 h post-ingestion. CONCLUSION: The presence of intact sCD14 in the blood and the gastrointestinal tract of newborns post-ingestion has implications in the development of allergies, obesity, and other inflammation-related pathogeneses later in life.

Human milk metagenome: a functional capacity analysis.

BACKGROUND: Human milk contains a diverse population of bacteria that likely influences colonization of the infant gastrointestinal tract. Recent studies, however, have been limited to characterization of this microbial community by 16S rRNA analysis. In the present study, a metagenomic approach using Illumina sequencing of a pooled milk sample (ten donors) was employed to determine the genera of bacteria and the types of bacterial open reading frames in human milk that may influence bacterial establishment and stability in this primal food matrix. The human milk metagenome was also compared to that of breast-fed and formula-fed infants' feces (n = 5, each) and mothers' feces (n = 3) at the phylum level and at a functional level using open reading frame abundance. Additionally, immune-modulatory bacterial-DNA motifs were also searched for within human milk. RESULTS: The bacterial community in human milk contained over 360 prokaryotic genera, with sequences aligning predominantly to the phyla of Proteobacteria (65%) and Firmicutes (34%), and the genera of Pseudomonas (61.1%), Staphylococcus (33.4%) and Streptococcus (0.5%). From assembled human milk-derived contigs, 30,128 open reading frames were annotated and assigned to functional categories. When compared to the metagenome of infants' and mothers' feces, the human milk metagenome was less diverse at the phylum level, and contained more open reading frames associated with nitrogen metabolism, membrane transport and stress response (P < 0.05). The human milk metagenome also contained a similar occurrence of immune-modulatory DNA motifs to that of infants' and mothers' fecal metagenomes. CONCLUSIONS: Our results further expand the complexity of the human milk metagenome and enforce the benefits of human milk ingestion on the microbial colonization of the infant gut and immunity. Discovery of immune-modulatory motifs in the metagenome of human milk indicates more exhaustive analyses of the functionality of the human milk metagenome are warranted.

Molecular strategies to engineer transgenic rice seed compartments for large-scale production of plant-made pharmaceuticals.

The use of plants as bioreactors for the large-scale production of recombinant proteins has emerged as an exciting area of research. The current shortages in protein therapeutics due to the capacity and economic bottlenecks faced with modern protein production platforms (microbial, yeast, mammalian) has driven considerable attention towards molecular pharming. Utilizing plants for the large-scale production of recombinant proteins is estimated to be 2-10% the cost of microbial platforms, and up to 1,000-fold more cost effective than mammalian platforms (Twyman et al. Trends Biotechnol 21:570-578, 2003; Sharma and Sharma, Biotechnol Adv 27:811-832, 2009). In order to achieve an economically feasible plant production host, protein expression and accumulation must be optimized. The seed, and more specifically the rice seed has emerged as an ideal candidate in molecular pharming due to its low protease activity, low water content, stable protein storage environment, relatively high biomass, and the molecular tools available for manipulation (Lau and Sun, Biotechnol Adv 27:1015-1022, 2009).

Indica rice cultivar IRGA 424, transformed with cry genes of B. thuringiensis, provided high resistance against Spodoptera frugiperda (Lepidoptera: Noctuidae).

Plant expression of the entomopathogenic bacteria Bacillus thuringiensis cry gene has reduced the damage created by insect pests in several economically important cultures. For this study, we have conducted genetic transformation of the indica rice "IRGA 424", via Agrobacterium tumefaciens, using the B. thuringiensis cry1Aa and cry1B genes, with the objective of obtaining rice plants resistant to the insect pests from this culture. The gene constructions harbor the promoters maize proteinase inhibitor and ubiquitin. The results showed that high concentration of the hormone 2,4-dichlorophenoxyacetic acid and agarose as the gelling agent helped the production of embryogenic calli for the analyzed cultivar. More than 80% of the obtained transformed plants revealed the integration, using polymerase chain reaction, of the cry1Aa and cry1B genes. Analysis of the expression of the heterologous protein by Western blotting revealed the expression of the Cry1B delta-endotoxin in IRGA 424 plants transformed with the ubiquitin promoter. Data showed the production and dissemination of a high number of embryogenic calli in addition to obtaining plants transformed with the cry1Aa and cry1B genes until the reproductive phase. The feed bioassays with the transformed plants and Spodoptera frugiperda (JE Smith) larvae indicated high rates of mortality to the insect target. The highest corrected mortality rate achieved under laboratory conditions with Bt-rice plants transformed with the cry1B and cry1Aa genes was 94 and 84%, respectively. Thus, our results demonstrated the great potential of transformed Bt-rice plants in controlling the damage caused by these insect pests in rice paddy fields.

Seed storage proteins of the globulin family are cleaved post-translationally in wheat embryos.

BACKGROUND: The 7S globulins are plant seed storage proteins that have been associated with the development of a number of human diseases, including peanut allergy. Immune reactivity to the wheat seed storage protein globulin-3 (Glo-3) has been associated with the development of the autoimmune disease type 1 diabetes in diabetes-prone rats and mice, as well as in a subset of human patients. FINDINGS: The present study characterized native wheat Glo-3 in salt-soluble wheat seed protein extracts. Glo-3-like peptides were observed primarily in the wheat embryo. Glo-3-like proteins varied significantly in their molecular masses and isoelectric points, as determined by two dimensional electrophoresis and immunoblotting with anti-Glo-3A antibodies. Five major polypeptide spots were identified by mass spectrometry and N-terminal sequencing as belonging to the Glo-3 family. CONCLUSIONS: These results in combination with our previous findings have allowed for the development of a hypothetical model of the post-translational events contributing to the wheat 7S globulin profile in mature wheat kernels.

Plant essential oils and mastitis disease: their potential inhibitory effects on pro-inflammatory cytokine production in response to bacteria related inflammation.

This paper highlights the role of plant volatile organic compounds, found in essential oils, for the treatment of bacteria related inflammation. This report is focused on tea tree oil, particularly its main compound terpinen-4-ol. Analysis of the published literature shows that many essential oils have significant antibacterial, antifungal and anti-inflammatory effects. Some of their major components, such as terpinen-4-ol, act by inhibiting pro-inflammatory cytokine expression while stimulating production of anti-inflammatory cytokines. Such observations may be exploited to encourage biotherapy against mastitis. The use of synthetic antibiotics is being increasingly discouraged because their presence in dairy milk may have potential downstream effects on population health and the agri-food chain. In the context of inflammation and related mammalian responses, understanding the interplay between volatile organic compounds, especially terpinen-4-ol, and cytokines during bacteria related inflammation should clarify their mode of action to control mastitis.

Strategy and tactics of disarming GHG at the source: N2O reductase crops.

Nitrous oxide (N(2)O), the third most abundant greenhouse gas (GHG), is highly stable and plays a significant role in stratospheric ozone destruction. The primary anthropogenic source of N(2)O stems from use of nitrogen fertilizers in soil. The bacterial enzyme nitrous oxide reductase (N(2)OR), naturally found in some soils, is the only known enzyme capable of catalyzing the final step of the denitrification pathway, conversion of N(2)O to N(2). In this opinion, we discuss potential biology-based strategies to reduce N(2)O by amplifying the amount of available enzyme catalyst in agri-system environments during crop growth and in post-harvest detritus. N(2)OR from Pseudomonas stutzeri has been tested in transgenic plants with promising results. Such seed-borne phytoremediation systems targeted towards GHGs merit field testing.

Expression of nitrous oxide reductase from Pseudomonas stutzeri in transgenic tobacco roots using the root-specific rolD promoter from Agrobacterium rhizogenes.

The nitrous oxide (N(2)O) reduction pathway from a soil bacterium, Pseudomonas stutzeri, was engineered in plants to reduce N(2)O emissions. As a proof of principle, transgenic plants expressing nitrous oxide reductase (N(2)OR) from P. stutzeri, encoded by the nosZ gene, and other transgenic plants expressing N(2)OR along with the more complete operon from P. stutzeri, encoded by nosFLZDY, were generated. Gene constructs were engineered under the control of a root-specific promoter and with a secretion signal peptide. Expression and rhizosecretion of the transgene protein were achieved, and N(2)OR from transgenic Nicotiana tabacum proved functional using the methyl viologen assay. Transgenic plant line 1.10 showed the highest specific activity of 16.7 µmol N(2)O reduced min(-1) g(-1) root protein. Another event, plant line 1.9, also demonstrated high specific activity of N(2)OR, 13.2 µmol N(2)O reduced min(-1) g(-1) root protein. The availability now of these transgenic seed stocks may enable canopy studies in field test plots to monitor whole rhizosphere N flux. By incorporating one bacterial gene into genetically modified organism (GMO) crops (e.g., cotton, corn, and soybean) in this way, it may be possible to reduce the atmospheric concentration of N(2)O that has continued to increase linearly (about 0.26% year(-1)) over the past half-century.

Expression of the nos operon proteins from Pseudomonas stutzeri in transgenic plants to assemble nitrous oxide reductase.

Nitrous oxide (N(2)O) is a stable greenhouse gas that plays a significant role in the destruction of the ozone layer. Soils are a significant source of atmospheric N(2)O. It is important to explore some innovative and effective biology-based strategies for N(2)O mitigation. The enzyme nitrous oxide reductase (N(2)OR), naturally found in soil bacteria, is responsible for catalysing the final step of the denitrification pathway, conversion of N(2)O to dintrogen gas (N(2)). To transfer this catalytic pathway from soil into plants and amplify the abundance of this essential mechanism (to reduce global warming), a mega-cassette of five coding sequences was assembled to produce transgenic plants heterologously expressing the bacterial nos operon in plant leaves. Both the single-gene transformants (nosZ) and the multi-gene transformants (nosFLZDY) produced active recombinant N(2)OR. Enzymatic activity was detected using the methyl viologen-linked enzyme assay, showing that extracts from both types of transgenic plants exhibited N(2)O-reducing activity. Remarkably, the single-gene strategy produced higher reductase capability than the whole-operon approach. The data indicate that bacterial N(2)OR expressed in plants could convert N(2)O into inert N(2) without involvement of other Nos proteins. Silencing by homologous signal sequences, or cryptic intracellular targeting are possible explanations for the low activities obtained. Expressing N(2)OR from Pseudomonas stutzeri in single-gene transgenic plants indicated that such ag-biotech solutions to climate change have the potential to be easily incorporated into existing genetically modified organism seed germplasm.

The starch granule associated proteomes of commercially purified starch reference materials from rice and maize.

Commercially available reference materials are integral components of many experimental protocols, as it is critical to compare one's results to those derived from well-characterized standards. Most reference materials are well defined, with all their components being cataloged. However, certain reference materials, such as commercially prepared starch samples, can have undefined components, potentially limiting their usefulness as standards. The proteome of commercially prepared starch has not been documented, and to that end, we initiated a mass spectrometry-based survey of the proteins associated with starch granules in commercially prepared rice and maize starch samples. We performed direct trypsin treatments of starch samples and sequenced both the water-soluble peptides liberated into the aqueous supernatant and the peptides released from the starch granule surface by isopropanol solvent washing. We discovered that the majority of proteins, in both rice and maize samples, were involved in either carbohydrate metabolism or storage. We also documented proteins that are markers for seed maturity and for starch mobilization.

Maternal separation and gastrointestinal transit time in neonate rats.

Gastrointestinal transit times (GItts) were compared in separate litters of 10- and 15-day-old Sprague Dawley rats using barium sulphate. By tracking the leading front of the bolus on radiographs, the gastrocaecal transit times in pups were estimated. To measure the total GItt, the duration from orogastric gavage until an observable defecation of barium sulphate was recorded. The gastrocaecal times for 10-day-old pups maintained with their dam (n = 5) ranged from 4-5 h and those removed from the dam ranged from 2.5-5 h. For 15-day-old pups with their dam (n = 6) and without dam (n = 5), gastrocaecal times ranged from 4-6 h and 3.5-5 h, respectively. Ten-day-old pups that remained with the dam had a GItt of 13.8 ± 0.9 h and those kept in the absence of the dam had a time of 9.3 ± 0.7 h. This decrease (P < 0.05) in GItt in the absence of the dam was age-dependent in 10-day-old pups, and was not observed (P > 0.05) in 15-day-old pups. The results provide a basis, for the design of future studies involving neonate rat metabolism, to include maternal presence.

(14)C radiolabeling of proteins to monitor biodistribution of ingested proteins.

The economical preparation of microgram quantities of (14)C-labeled proteins by in vacuo methylation with methyl iodide is described. The (14)C radiolabeling was achieved by the covalent attachment of [(14)C]methyl groups onto amino and imidazole groups by reaction in vacuo with [(14)C]methyl iodide. The method was tested by investigating the biodistribution of (14)C in rats that were fed (14)C-labeled human soluble cluster of differentiation 14 (CD14) protein, a receptor for bacterial lipopolysaccharide. Two other control proteins, bovine serum albumin (BSA) and casein, were also labeled with (14)C and used for comparative analysis to determine the following: (i) the efficacy and cost efficiency of the in vacuo radiolabeling procedure and (ii) the extent of incorporation of the (14)C label into the organs of orogastrically fed 10-day-old Sprague-Dawley rats. [(14)C]BSA, [(14)C]casein, and [(14)C]CD14 were individually prepared with specific radioactivities of 34,400, 18,800, and 163,000 disintegrations per minute (dpm)/microg, respectively. It was found that the accumulation of (14)C label in the organs of [(14)C]CD14-fed rats, most notably the persistence of (14)C in the stomach 480 min postgavage, was temporally and spatially distinct from [(14)C]BSA and [(14)C]casein-fed rats.